Ticks and tick‐borne pathogens in selected abattoirs and a slaughter slab in Kumasi, Ghana

Abstract Background Ticks are vectors of pathogens that affect the health of animals and humans. With the constant trade of livestock across borders, there is the risk of new tick species invasion accompanied by the spread of infectious tick‐borne pathogens. Aim This study sought to determine the diversity of tick species within abattoirs and a slaughter slab as well as identify the pathogens carried by these ticks. Methods The ticks were collected from slaughtered cattle, identified and screened for pathogens using PCR and sequencing. Results A total of 371 ticks were collected from slaughtered cattle across the three sampling sites: Kumasi abattoir (288, 77.63%), Akwatia Line slaughter slab (52, 14.02%) and Suame abattoir (31, 8.35%). The predominant species was Amblyomma variegatum (85.44%) with Rhipicephalus sanguineus (s.l.) (0.27%) as the least occurring species. Total nucleic acid from the tick pools was screened for pathogens based on the nucleoprotein gene region in the S segment of the Crimean–Congo haemorrhagic fever virus (CCHFV) genome, the 295‐bp fragment of the transposase gene of the Coxiella burnetii IS1111a element, the 560 bp segment of the ssrRNA gene of Babesia and Theileria, the 345 bp fragment of the Ehrlichia genus 16SrRNA gene and the rOmpA gene (OmpA) of Rickettsia. From the 52 tick pools screened, 40 (76.92%) were found positive for pathogen DNA. The pathogens identified were Rickettsia africae (69.23%), Rickettsia aeschlimannii (7.69%), C. burnetii (5.77%), uncultured Ehrlichia sp. (5.77%), Candidatus Midichloria mitochondrii (3.85%) and CCHFV (3.85%). A significant association was observed among A. variegatum, Hyalomma rufipes, Hyalomma truncatum and occurring tick‐borne pathogens R. africae, R. aeschlimannii and uncultured Ehrlichia sp. (p < 0.001). Conclusion The findings show the occurrence of zoonotic pathogens, suggesting an increased risk of infections among the abattoir workers. There is a need to adopt control measures within the abattoirs to prevent pathogen spread.


INTRODUCTION
Worldwide, about 80% of the cattle population, especially in tropical and subtropical countries, are significantly affected by tick infestation (Magesa et al., 2023).Tick infestation in cattle often results in direct consequences such as blood and weight loss, as well as indirect effects due to the role of ticks as vectors of infectious diseases (Sahara et al., 2019).Ticks are blood-feeding ectoparasites that belong to the order Ixodida and are known vectors of various zoonotic diseases worldwide (Eisen et al., 2016).Nearly 10% of the 900 species of known ticks are capable of transmitting infectious pathogens to animals and humans, making ticks significant vectors of numerous diseases that affect animals and humans (Jongejan & Uilenberg, 2004).
It has been reported that the preferred ecological settings of each tick species influence where tick-borne diseases are most likely to occur (Parola & Raoult, 2001).There are close to 50 indigenous tick species in Africa that are known to infect domestic and livestock animals.Amblyomma, Hyalomma and Rhipicephalus are three genera among these unique species that have the greatest impact on animal health (Reye et al., 2012).Humans get infected with tick-borne pathogens either through tick bites or coming into contact with an infected animal (Telmadarraiy et al., 2015).
The geographic spread and infection rates of zoonotic pathogens spread by ticks have risen, and they are anticipated to constitute a significant risk to human health in the future (Estrada-Peña & De La Fuente, 2014).In countries throughout Africa, the Middle East, the Balkans and Asia, the Crimean-Congo haemorrhagic fever virus (CCHFV), for example, causes severe outbreaks of viral haemorrhagic fever, characterized by a case fatality estimated to range from 10% to 40% (Shahhosseini et al., 2021).The zoonotic pathogen Coxiella burnetii is naturally present in more than 40 different tick species (Maurin & Raoult, 1999).Even though C. burnetii is transmitted through contact with infected animals or animal products, ticks can transmit infections as the bacteria are shed through their faeces (Angelakis & Raoult, 2010).Again, it has been observed that rickettsial infections have spread to non-endemic areas due to increasing tick bites (Parola et al., 2013).In Africa, tick species Amblyomma hebraeum or Amblyomma variegatum are primarily responsible for the spread of Rickettsia species depending on the geographical location (Parola et al., 2005).
Limited studies conducted in Ghana indicate the occurrence of tick species and tick-borne pathogens.Among these pathogens are Rickettsia spp., C. burnetii (Nimo-Paintsil et al., 2022), CCHFV (Akuffo et al., 2016), Anaplasma spp.(Addo, Olivia, et al., 2023) and Babesia spp.(Bell-Sakyi et al., 2004;Nagano et al., 2013), all of which have been identified in Ghana.These pathogens pose a threat to both animals and humans; hence, there is a need to prevent their spread.Abattoir employees face a heightened risk of zoonotic infections due to their direct contact with livestock.Unfortunately, many of these infections go unnoticed, unreported and misdiagnosed as a result of limited resources.Furthermore, animals can serve as reservoirs and amplifying hosts for zoonotic pathogens.This situation has been reported in the Upper East region of Ghana, where livestock from the abattoir were found to harbour zoonotic tick-borne pathogens (Addo, Bentil, Yartey, et al., 2023).
Given these factors, this study sought to identify tick species infesting slaughtered cattle in Kumasi and determine the presence of tick-borne pathogens that could potentially infect the abattoir workers.

METHODS
The study was conducted in two different abattoirs and a slaughter slab within the Kumasi municipality of Ghana (Figure 1).The study sites were the Akwatia Line slaughter slab at Amakom, the Kumasi abattoir at Kaase and the Suame abattoir, where livestock from within the community and beyond are slaughtered daily.On average, the cattle slaughtered daily are about 200 in the Kumasi abattoir, 25 in the Akwatia Line slaughter slab and 30 in the Suame abattoir.After verbal consent, ticks were manually removed post-slaughter from the cattle and placed in labelled 15 mL falcon tubes.The health status of the cattle could not be confirmed as they were slaughtered before the tick collections were done.The majority of the ticks were unfed; a few were partially engorged, and a lesser number were fully engorged.The ticks were sent to the laboratory where they were morphologically identified using taxonomic keys (Walker et al., 2003), pooled (1-10) per animal according to the specific species, sex and location and kept in 2 mL Eppendorf tubes containing RNAlater for storage at −80 • C until molecular analysis.

TICK-BORNE PATHOGEN DETECTION AND ANALYSIS
Total nucleic acid was extracted from each tick pool using the QIAamp Mini Kit following the manufacturer's instructions (Crowder et al., 2010).The presence of CCHFV was determined using an assay that amplifies the conserved region of the CCHFV genome, specifically the F I G U R E 1 Map showing the tick collection sites.nucleoprotein gene region in the S segment (Atkinson et al., 2012).Furthermore, C. burnetii was detected using an assay that amplifies the 295-bp fragment of the transposase gene of the C. burnetii IS1111a element (Klee et al., 2006).Samples with cycle threshold (Ct) values below 40 were deemed positive for CCHFV or C. burnetii after comparing their curves to those of the positive control.A 632 bp amplification was achieved using primers that target the rOmpA gene (OmpA) of Rickettsia to detect the presence of Rickettsia DNA in tick pools (Jiang et al., 2005).The occurrence of Babesia/Theileria DNA was detected using primers that target the 560 bp segment of the ssrRNA gene of Babesia and Theileria (Beck et al., 2009).Using primers that target the 345 bp fragment of the Ehrlichia genus 16SrRNA gene (Nazari et  The pathogens from this investigation were subjected to sequence alignments and phylogenetic analysis using MEGA X (Kumar et al., 2018).The neighbour-joining method was used to construct the phylogenetic tree.Thousand bootstrap replicates were used to calculate the confidence indices inside the phylogenetic trees, and the findings were shown on the branches as percentages.For the GenBank sequences used in the phylogenetic analysis, the different accession numbers and countries of origin have been mentioned.

Statistical analysis
The statistical analysis was carried out using STATA version 13, and the level of significance was set at p < 0.05.The chi-square test was performed to assess the association between the occurrence of the detected pathogens and factors such as tick species and location.
We again assessed the pooled prevalence (minimum infection) of the different pathogens by location and species using proportions with exact confidence intervals.We then assessed the maximum likelihood estimation (MLE) using methods described in the PooledInfRate R package.All analysis was conducted in R version 4.3.3.

PATHOGENS IDENTIFIED
From the 52 tick pools screened, 40 (76.92%) were found positive for pathogen DNA.Generally, the pathogens identified were Rickettsia TA B L E 2 Association among the identified pathogens, tick species and location.

DISCUSSION
In this study, ticks were collected from cattle across three sites in tick species in this study were A. variegatum, which has been reported as widely distributed in Ghana (Bell-Sakyi et al., 1996;Walker & Koney, 1999) and occurs in more than 30 African countries (Walker et al., 2003).A. variegatum can cause severe damage to the livestock industry by reducing milk production, impairing animal growth and transmitting Ehrlichia ruminantium (Esemu et al., 2013;Stachurski, 2000).In Ghana, C. burnetii, Rickettsia species and CCHFV have been detected in A. variegatum, indicating its public health importance and a need for enforcing control measures (Addo, Bentil, Baako, et al., 2023;Akuffo et al., 2016;Nimo-Paintsil et al., 2022).It should also be noted that other tick species identified in the study sites are capable of spreading various pathogens of veterinary and zoonotic importance (Reye et al., 2012).The Hyalomma species identified in this study can transmit diverse pathogens, including CCHFV in humans (Aktas et al., 2014;Gargili et al., 2017) and Theileria annulata in cattle (Mamman et al., 2020).R. sanguineus (s.l.) was the least tick species collected from the Kumai abattoir.This species is also known as the brown dog tick with a preference for dogs as hosts (Dantas-Torres, 2008) but can be found infesting cattle at low numbers.Nonetheless, R. sanguineus (s.l.) can spread pathogens, such as Ehrlichia, Babesia and Rickettsia (Harrus et al., 1999;Tavassoli et al., 2013;Wikswo et al., 2007).Understanding the tick population dynamics and species prevalence is essential for developing effective tick control and management strategies, ultimately benefiting the health and well-being of livestock and, consequently, the agricultural industry.
R. africae was detected mostly in A. variegatum and, to a lesser extent, H. truncatum in this study.R. africae is responsible for the disease African tick-borne fever (Kelly et al., 1996) and is the principal cause of fever in travellers returning from Africa (Mediannikov, Diatta, et al., 2010).The findings of this study can be compared to previous studies in Ghana (Addo, Bentil, Baako, et al., 2023;Nimo-Paintsil et al., 2022), Liberia and Guinea (Mediannikov et al., 2012) that found a high occurrence of Rickettsia DNA and R. africae in A. variegatum.There is an increased risk of R. africae transmission across the study sites, especially in light of its ability to be transmitted through transstadial and transovarial routes (Socolovschi et al., 2009) and the high occurrence of the principal vector A. variegatum (Parola et al., 2013).R. aeschlimannii was also identified in only H. rufipes in the Kumasi abattoir and Akwatia Line slaughter slab.This can be compared to studies in Ghana (Addo, Bentil, Baako, et al., 2023) and Nigeria (Kamani et al., 2015) that found H. rufipes infected with R. aeschlimannii.The findings show that different Rickettsia species occur in Ghana, and there is a need to fully determine the circulating species to create effective control strategies.C. burnetii was also identified in pools of A. variegatum in the Kumasi abattoir.This can be compared to previous studies in Ghana (Addo, Bentil, Baako, et al., 2023;Nimo-Paintsil et al., 2022) and Senegal (Mediannikov, Fenollar, et al., 2010) that found ticks infected with C.
burnetii.Reports from studies and the findings from this study suggest that ticks could play a role in the transmission of this pathogen.
CCHFV is an infectious pathogen transmitted primarily by species of the genera Hyalomma, although other tick species can transmit the virus (Spengler & Estrada-Peña, 2018).In this study, A. variegatum from the Kumasi abattoir was found infected with the virus.This can be compared to a previous study in the same abattoir which found ticks, including A. variegatum, to be infected with CCHFV and pathogen exposure among the abattoir workers (Akuffo et al., 2016).The virus causes asymptomatic infections in wild and domestic animals (Bannazadeh & Aghazadeh, 2016;Fatemian et al., 2018) but leads to clinical symptoms such as haemorrhage and cardiovascular and neuropsychiatric abnormalities in humans (Whitehouse, 2004).Abattoir workers are at risk of infections; hence, there is a need to adopt effective preventive and control measures.A study in Ghana has reported the occurrence of Ehrlichia canis and Ehrlichia minasensis in ticks (Addo, Olivia, et al., 2023).
In this study, uncultured Ehrlichia sp. was detected in H. truncatum from the Kumasi abattoir.This means that different Ehrlichia species are in circulation in Ghana.To develop an effective control measure, it will be necessary to conduct more surveillance activities to determine the species in circulation and their effects on livestock production.Ca.M.
mitochondrii is a bacterial endosymbiont of ticks that is found inside the mitochondria and occurs frequently in Ixodes ricinus (Stavru et al., 2020).In this study, it was detected in H. rufipes in the Kumasi abattoir.It is not clear if this symbiont influences pathogen transmission, although a study reported a positive correlation between varied levels of Midichloria mitochondrii and the occurrence of pathogen Rickettsia parkeri (Budachetri et al., 2018).The possibility of simultaneous transmission of several infections to hosts, including humans and animals, is raised by the co-occurrence of numerous pathogens in the same tick species.This study found coinfections of R. africae and CCHFV in A. variegatum as well as R. africae and uncultured Ehrlichia sp. in H.
truncatum.Co-feeding on the same host allows for the exchange of pathogens between tick species and the animal host, increasing the risk of pathogen transmission.

CONCLUSION
In this study, A. variegatum was the predominant tick species and was found infected with multiple zoonotic pathogen DNA, including R. africae, C. burnetii and CCHFV.The majority of these pathogens were identified in the Kumasi abattoir indicating an increased risk of infection to the abattoir workers in direct contact with the cattle.The findings highlight the necessity of tick management, surveillance and preventative efforts to safeguard human and animal populations from tick-borne pathogens of public health and veterinary importance.
Kumasi.The majority of the ticks were collected from the Kumasi abattoir due to the large number of cattle slaughtered daily compared to the Akwatia Line slaughter slab or Suame abattoir.The predominant F I G U R E 2 Phylogenetic tree of Rickettsia species based on the OmpA gene.The sequences from this study are indicated as S14, S16 and S19.F I G U R E 3 Phylogenetic tree of Anaplasma and Ehrlichia based on the 16SrRNA gene.The sequences obtained in this study are indicated as S7 and S8.
Distribution of tick-borne pathogens in the identified tick species.
al., TA B L E 1 Abbreviation: CCHFV, Crimean-Congo haemorrhagic fever virus.2013), the presence of Ehrlichia and Anaplasma DNA was confirmed in the tick pools.The PCR products from the conventional PCRs were separated on 2% agarose gel, visualized using a Molecular Imager Gel Doc and positive products were purified and sequenced by Macrogen Europe B.V.